De novo CNV formation in vivo with sickle cell anemia therapy

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DESCRIPTION (provided by applicant): Sickle cell disease (SCD) is the first molecular genetic disorder identified in humans, affecting over 50,000 Americans and millions of people worldwide. Hydroxyurea (HU) is currently the only approved disease- modifying therapy for adult SCD and is in late-stage clinical trials for treatment of affected infants and children. HU stimulates fetal hemoglobin production and is therefore effective in improving oxygen transport while reducing the incidence and severity of vaso-occlusive crises. However, HU is also a known replication inhibitor, mutagen and clastogen, yet the genetic effects of HU in the offspring and grandchildren of treated subjects have not been studied, particularly at the genomic level for effects of replication stress such as copy number variant (CNV) mutations. CNVs are a key factor in normal genetic variation and evolution and are a common and important class of mutation in genetic disorders, including mental retardation, autism, schizophrenia and many others. We have developed a human cell culture model system to investigate the genetic and environmental risk factors for CNV mutations. We have found that HU, at concentrations identical to serum concentrations in treated patients, significantly induces CNVs in normal human cells. These findings have important and direct genetic implications for deleterious, de novo CNV risk in the children and grandchildren of patients treated with HU for SCD and other disorders. We propose to extend these findings to direct studies in animal models in vivo. We will evaluate the genetic effects of HU on CNV induction in both the male and female germlines by examining F1 and F2 generations of treated mice using high-resolution genomic microarrays. We will determine parental origins of de novo CNVs, examine their genomic structures to infer cellular and mechanistic origins and compare findings with those from phenotyping of study group animals. In addition, we will assess somatic mosaicism for CNVs in cells and tissues of mice treated prenatally with HU and controls. These studies will provide the first in vivo test in mammals of the somatic mutation/replication stress hypothesis for CNVs using a model inhibitor of replication. The results obtained will have important implications for defining genetic and environmental risk factors for both germline and somatic deleterious CNVs in humans. Moreover they will have important and immediate clinical significance to a large number of individuals with SCD and the future generations of HU-treated patients.